A conventional cell electrophysiological measurement device as an example of a sensor device can allow a cell as a biomaterial substance to be adsorbed on a surface of a sensor chip and measure the property of the cell. The patch-clamp method in electrophysiology is known as a method for measuring an ion channel that is present in a cell membrane. The patch-clamp method has elucidated various functions of the ion channel. The functions of the ion channel are important interest in the cytology, and are applied for developing agents.
On the other hand, however, the patch-clamp method requires an operation based on extremely advanced technique of inserting a minute micropipette into a single cell with high accuracy. Therefore, even a skilled operator cannot carry out much measurement. Therefore, the patch-clamp method is not so suitable when measurement with high throughput is required.
Thus, flat-shaped cell electrophysiological measurement devices using a fine processing technique have been developed. These devices are suitable for an automated patch system that does not require insertion of a micropipette into individual cells.
A cell electrophysiological measurement device includes, for example, a mount board made of resin, and a sensor chip inserted into a communicating hole provided on a bottom surface of the mount board. The sensor chip is made of silicon or glass. Furthermore, the sensor chip includes a curved or flat surface partition plate provided with at least one or more through holes. The opening diameter of the through hole is 1 micrometer to 3 micrometers. The cell electrophysiological measurement device includes a single-hole sensor chip having one through hole in one sensor chip, and a multi-hole sensor chip having a plurality of through holes in one sensor chip.
An upper electrolytic chamber disposed on the upper side of the partition plate of the mount board and a lower electrolytic chamber disposed on the lower side of the partition plate of the mount board are filled with an electrolytic solution. The upper electrolytic chamber and the lower electrolytic chamber are divided by the partition plate included in the mount board and the sensor chip.
Then, a cell as a specimen is injected from the upper electrolytic chamber, the cell is adsorbed on an opening at the upper electrolytic chamber side of the through hole by pressurizing from the upper electrolytic chamber or sucking from the lower electrolytic chamber. The opening diameter of the through hole is 1 micrometer to 3 micrometers. This range is suitable for holding and adsorbing a cell. A part of a sucked cell membrane is extended to the inside of the through hole and fixedly attached to a wall surface of the through hole.
In this state, for example, an agent is administered to the periphery of the cell, and potential difference or an electric current generated between the upper and lower electrolytic chambers is measured by using electrodes each of which is disposed inside the upper and lower electrolytic chambers, and thereby a pharmacological reaction of the cell with respect to the administered agent can be determined (PTL 1).
It is more difficult to establish a high-resistance seal between the sensor chip and a cell in a conventional flat-shaped cell electrophysiological measurement device as compared with the patch-clamp method using a glass pipette. Herein, a high-resistance state in which the sensor chip and a cell are fixedly attached to each other strongly is referred to as a gigaseal state.
Furthermore, a plane (a cell contact plane) which a cell is brought into contact with and fixedly attached to is a partition plate and a wall surface of a through hole. In order to increase the degree of adhesion of a cell and to form strong fixedly-attached state, a silanol group (SiOH group) is required to be sufficiently formed on the surface of the cell contact plane. An exemplary embodiment in which the degree of adhesion between a cell contact plane of a glass pipette and a cell is increased is disclosed (NPTL 1). Furthermore, there is also a disclosure that surface energy and a hydrophilic property are changed depending upon the amount of silanol groups attached on the surface of glass or SiO2 (NPTL 2). That is to say, in order to fixedly attach a cell to the cell contact plane strongly, a sufficient amount of silanol groups is required to be attached. The amount of silanol groups can be indirectly evaluated by the hydrophilic property of the surface, a surface charge, or the like.
Examples of methods for improving the hydrophilic property of the surface of glass or SiO2 include a method using plasma such as oxygen plasma and atmospheric plasma, a method of irradiation with light with high energy, for example, excimer UV right exposure, a method of washing with a mixture solution of sulfuric acid and a hydrogen peroxide solution, and the like. In this way, methods commonly used in production of semiconductors or production of MEMS devices can be employed (NPTL 3).
Furthermore, in order to form a gigaseal state, a method for enhancing an activity by subjecting a silicon dioxide layer on the surface of the chip surface to treatment with, for example, oxygen plasma may be employed. For example, there is a disclosure that a chip surface is subjected to acid/base treatment, oxygen plasma treatment, or the like, thereby forming SiOM (M represents H or metal such as Na, K, Mg, and Ca) (NPTL 2).
Alternatively, film formation of a silicon nitride layer containing Si3N4 as a main component on a surface of a chip by using a LPCVD (Low Pressure Chemical Vapor Diposition) method is disclosed (PTLs 3 and 4).
In conventional sensor devices, when measurement is carried out in a short time after a sensor device is produced, measurement can be carried out with an amount of silanol groups formed on the surface kept sufficient. However, when it is necessary to store a large number of sensor devices produced simultaneously for a considerable time before measurement is started, it is necessary to store the sensor devices while the silanol groups formed on the surface are kept in a sufficient amount. In a conventional sensor device, for example, in a storage state before measurement is carried out by inputting a cell, a silanol group on the surface of the sensor device is dissociated so as to form Si.O.Si (a siloxane bond). Alternatively, a substance that inhibits adsorption of a cell to be measured (hereinafter, referred to as an “adsorption inhibiting substance”) is adsorbed on the surface of the sensor device, which may make attachment of the cell insufficient and reduce the accuracy of measurement.
That is to say, in a conventional cell electrophysiological measurement device as one example of a sensor device, a cell is not sufficiently adsorbed on the through hole because a cell adsorption inhibiting substance is present on a surface of a through hole on which the cell formed on a sensor chip is to be adsorbed. Therefore, a leakage current may be generated between the cell surface and the through hole surface, so that it is difficult to improve the measurement accuracy. Therefore, as means for preventing of silanol groups from being dissociated and means for preventing adsorption inhibiting substances from being adsorbed on the surface of through hole, underwater storage for storing a sensor device in water, vacuum storage for storing a sensor device in a vacuum chamber, and inert gas storage for storing a sensor device in an atmosphere of an inert gas such as N2, immediately before a cell is measured. However, the underwater storage requires work for pulling water out of the sensor before measurement, and the vacuum storage or the inert gas storage may be insufficient in terms of a storage effect.
In particular, the dissociation of silanol groups advances a mechanism for forming H2O in which, as shown in the chemical formula Chem. 1, two silanol groups adjacent to each other on the surface are dissociated and bound. Therefore, the vacuum storage or the inert gas storage may have an effect for preventing attachment of adsorption inhibiting substances, dissociation of silanol groups and generation of H2O cannot be prevented. Therefore, in the vacuum storage and the inert gas storage, an effect for preventing deterioration of the hydrophilic property is limited.2SiOH⇄Si.O.Si+H2O  [Chem. 1]
Note here that examples of prior art mentioned above include the following patent literatures and non-patent literatures.